CLINICAL CASE STUDYpublished: 23 March 2015

doi: 10.3389/fgene.2015.00102

Frontiers in Genetics | www.frontiersin.org 1 March 2015 | Volume 6 | Article 102

Edited by:

Jumana Al-Aama,

King Abdulaziz University, Saudi Arabia

Reviewed by:

Aída Beatriz Falcón De Vargas,

Universidad Central de Venezuela,

Venezuela

Veronika Boczonadi,

Newcastle University, UK

*Correspondence:

Sara Brito,

Serviço de Pediatria, Centro

Hospitalar de Leiria, Hospital de Santo

André, Rua das Olhalvas, 2410-197

Leiria, Portugal

sarabri@gmail.com

Specialty section:

This article was submitted to Genetic

Disorders, a section of the journal

Frontiers in Genetics

Received: 18 December 2014

Accepted: 26 February 2015

Published: 23 March 2015

Citation:

Brito S, Thompson K, Campistol J,

Colomer J, Hardy SA, He L,

Fernández-Marmiesse A, Palacios L,

Jou C, Jiménez-Mallebrera C,

Armstrong J, Montero R, Artuch R,

Tischner C, Wenz T, McFarland R and

Taylor RW (2015) Long-term survival

in a child with severe encephalopathy,

multiple respiratory chain deficiency

and GFM1 mutations.

Front. Genet. 6:102.

doi: 10.3389/fgene.2015.00102

Long-term survival in a child withsevere encephalopathy, multiplerespiratory chain deficiency andGFM1 mutationsSara Brito 1, 2*, Kyle Thompson 3, Jaume Campistol 2, 4, Jaime Colomer 2, Steven A. Hardy 3,

Langping He 3, Ana Fernández-Marmiesse 5, Lourdes Palacios 6, Cristina Jou 7,

Cecilia Jiménez-Mallebrera 2, 4, Judith Armstrong 4, 8, Raquel Montero 4, 9, Rafael Artuch 4, 9,

Christin Tischner 10, Tina Wenz 10, 11, Robert McFarland 3 and Robert W. Taylor 3

1 Serviço de Pediatria, Centro Hospitalar de Leiria, Hospital de Santo André, Leiria, Portugal, 2Neuromuscular Unit,

Neuropaediatrics Department, Hospital Sant Joan de Déu, Barcelona, Spain, 3Wellcome Trust Centre for Mitochondrial

Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK, 4Centro de Investigación Biomédica

en Red de Enfermedades Raras, Instituto de Salud Carlos III, Barcelona, Spain, 5Diagnosis and Treatment Unit for Inborn

Errors of Metabolism, Hospital Clínico Universitario de Santiago de Compostela, La Coruña, Spain, 6 Progenika Biopharma a

Grifols Company, Derio, Spain, 7 Pathology Department, Hospital Sant Joan de Déu, Esplugues Barcelona, Spain,8 Biochemical, Genetics and Rett Unit, Laboratory Department, Hospital Sant Joan de Déu, Esplugues Barcelona, Spain,9 Biochemical Department, Hospital Sant Joan de Déu, Esplugues Barcelona, Spain, 10Cluster of Excellence: Cellular Stress

Responses in Aging-Associated Diseases (CECAD), Institute for Genetics, University of Cologne, Cologne, Germany,11German Network for Mitochondrial Disorders (mitoNET), Munich, Germany

Background: Mitochondrial diseases due to deficiencies in the mitochondrial oxidative

phosphorylation system (OXPHOS) can be associated with nuclear genes involved

in mitochondrial translation, causing heterogeneous early onset and often fatal

phenotypes.

Case report: The authors describe the clinical features and diagnostic workup of

an infant who presented with an early onset severe encephalopathy, spastic-dystonic

tetraparesis, failure to thrive, seizures and persistent lactic acidemia. Brain imaging

revealed thinning of the corpus callosum and diffuse alteration of white matter signal.

Genetic investigation confirmed two novel mutations in the GFM1 gene, encoding

the mitochondrial translation elongation factor G1 (mtEFG1), resulting in combined

deficiencies of OXPHOS.

Discussion: The patient shares multiple clinical, laboratory and radiological similarities

with the 11 reported patients with mutations involving this gene, but presents with

a stable clinical course without metabolic decompensations, rather than a rapidly

progressive fatal course. Defects inGFM1 gene confer high susceptibility to neurologic or

hepatic dysfunction and this is, to the best of our knowledge, the first described patient

who has survived beyond early childhood. Reporting of such cases is essential so as

to delineate the key clinical and neuroradiological features of this disease and provide a

more comprehensive view of its prognosis.

Keywords: GFM1, mtEFG1, mitochondrial disorders, brain MRI, encephalopathy

Brito et al. Long surviving GFM1 mutation patient

Introduction

Mitochondrial diseases due to deficiencies in the mitochondrialoxidative phosphorylation system (OXPHOS) have an estimatedbirth prevalence of 1 in 10,000 (Schaefer et al., 2008). Mito-chondria are semi-autonomous organelles. They contain theirown genome (mtDNA), but rely on the nuclear DNA for fullfunctionality. The nuclear genome encodes approximately 1500mitochondrial proteins (Elstner et al., 2008) and provides essen-tial factors for mtDNA replication, transcription, translation, andassembly of the five OXPHOS complexes. Human mtDNA is aminimal genome and encodes only 13 essential subunits of theOXPHOS system as well as 2 rRNAs and 22 tRNAs (Andersonet al., 1981). Mitochondria possess their own translation systemto synthesize the mtDNA-encoded proteins. With the exceptionof complex II, all OXPHOS enzymes derive from dual geneticorigin with at least one subunit encoded by the mtDNA, thus,mitochondrial protein synthesis is crucial for OXPHOS func-tion and ATP generation. It is therefore not surprising, thatdefects in the mitochondrial translation machinery are an emerg-ing and increasingly recognized group of mitochondrial disor-ders (Jacobs and Turnbull, 2005; Scaglia and Wong, 2008; Smitset al., 2010; Kemp et al., 2011). Defects in mitochondrial proteintranslation are hypothesized to be the primary cause of com-bined OXPHOS deficiencies, in which the activity of more thanone OXPHOS complex is compromised (Smits et al., 2010; Kempet al., 2011; Rotig, 2011). New sequencing technologies haverapidly increased the canon of genes known to cause combinedrespiratory chain deficiency, an effect largely exerted through dis-ruptedmitochondrial DNAmaintenance, nucleotide transport orvarious aspects of translation including tRNA modification andaminoacylation (Taylor et al., 2014).

The early genetic investigation involving patients with defec-tive mitochondrial translation led to the description of a numberof nuclear-encoded defects in initiation, elongation, and termi-nation factors required for mitochondrial translation (Ylikallioand Suomalainen, 2012). These seem to be responsible for hetero-geneous phenotypes, manifesting predominantly in tissues withhigh energy requirements and with early onset and rapidly pro-gressive fatal courses (Antonicka et al., 2006; Balasubramaniamet al., 2012).

Here we describe the clinical features and diagnosticworkup of an infant who presented with an early onsetsevere encephalopathy resulting from combined deficiencies ofOXPHOS due to recessive mutations (including one novel vari-ant) in a nuclear gene (GFM1), encoding mitochondrial transla-tion elongation factor G1 (mtEFG1) and compare these findingswith other reported cases.

Background

Case ReportThis female patient (Figure 1A), currently 5 years 6 months old,was born to unrelated Caucasian parents following a pregnancythat was uneventful until 36 weeks gestation, when acute oligohy-dramnios and late presenting breech were diagnosed. Cesareansection was performed without complication and no neonatal

resuscitation was required. Birth weight was on the 50th cen-tile, length on the 10th and head circumference on the 97thcentile. Neonatal metabolic and hearing screenings were nor-mal. At 2 months old, she developed paroxysms of irritabilitythat evolved into a persistently irritable state with inconsolablecrying, opisthotonic posturing, feeding difficulties, and failureto thrive. At presentation, she had severe axial hypotonia withpoor head control, spasticity of extremities and severely impairedvisual tracking; there were no noticeable dysmorphic features.At 7 months old, she was already developmentally delayed priorto the onset of infantile spasms, which showed prompt clini-cal resolution with medication, although electrical abnormalitiespersisted on the electroencephalogram. She gained eye contactand social smile, but has never been able to sit independentlyand has not acquired verbal language skills. She has developed a

FIGURE 1 | Brain magnetic resonance imaging. (A) Photograph of the

patient aged 5 years demonstrating left convergent squint and normal facial

appearance. (B) Axial T2-weighted image at 29 months, revealing enlarged

lateral ventricles. (C) Coronal T2-FLAIR image at 29 months, displaying

abnormal white matter signal in a periventricular distribution. (D) Midsagittal

T1-FLAIR image at 29 months, showing thinning of the corpus callosum with

enlarged subarachnoid spaces. (E) Coronal T2-FLAIR image at 5 years 6

months, showing abnormal periatrial signal and enlarged lateral ventricles. (F)

Axial 3D FSPGR image at 5 years 6 months, displaying abnormal signal in the

posterolateral region of the putamen nucleus.

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Brito et al. Long surviving GFM1 mutation patient

spastic-dystonic tetraparesis with dystonic movements affectingher face, particularly her mouth.

After excluding perinatal metabolic and infectious causes, fur-ther investigation confirmed persistently elevated lactate levels inblood (maximum 4.87mmol/L; normal range 0.77–2.44mmol/L)and cerebral spinal fluid (CSF; 4.79mmol/L; normal range1.11–2.22mmol/L), along with elevated blood pyruvate (maxi-mum 0.15mmol/L; normal range 0.03–0.10mmol/L) and alanine(maximum 742 umol/L; normal range 226–416 umol/L). Therewas no hepatic dysfunction. The remaining blood, CSF and urineanalysis were unremarkable.

Brain magnetic resonance revealed widened subarachnoidspaces, Sylvian sulcus and ventricular system; diffuse and non-specific alteration of white matter signal and thinning of the cor-pus callosum (Figures 1B–D). These findings were only slowlyprogressive and apparently unchanged on repeat cranial MRimaging at 16 and 29 months old. Spectroscopy showed no sig-nificant changes for age, including lactate. However, repeat MRIperformed at 5 years 6 months old did show some further ven-tricular enlargement (Figure 1E) and abnormal signal change inthe posterolateral putamen (Figure 1F).

Electroencephalography showed a poorly formed backgroundwith multifocal polyspike paroxysms. Visual evoked potentialsrevealed delayed conduction and brainstem auditory evokedpotentials indicated bilateral vestibulocochlear nerve involve-ment. Electromyography demonstrated discrete involvement ofthe lower limb motor axons without demyelination. Muscle andskin biopsies were performed to investigate a likely mitochon-drial disorder.

Materials and MethodsAll procedures followed were in accordance with the ethical stan-dards of the responsible committee on human experimentation(institutional and national) and with the Helsinki Declaration of1975, as revised in 2000. Informed consent was obtained from theparents of the patient, including consent about the publication ofidentifying information.

Histopathological and Biochemical StudiesA diagnostic muscle biopsy (quadriceps) was orientated, frozenand processed for histology and histochemistry following stan-dard procedures. Respiratory chain complex activities weremeasured and normalized to citrate synthase activity incultured fibroblasts as previously described (Kirby et al.,2007).

Genetic Studies

Mitochondrial DNA analysisReal-time PCR analysis of mtDNA levels was undertaken in mus-cle DNA to investigate a possible mtDNA depletion syndrome.Southern blot analysis of mtDNA linearized following PvuIIdigestion was performed using a probe obtained by amplificationof a region of the human MT-RNR1 gene, whilst mtDNA rear-rangements were also screened by long-range PCR. The sequenceof the entire mitochondrial genome was determined in muscleusing standard protocols.

Next generation sequencing of nuclear-mitochondrial

genesSimultaneous sequencing of the coding regions (exons and exon-intron junctions) of 150 nuclear-encoded genes associated withmitochondrial respiratory chain defects was performed usingnext generation sequencing technology consisting of an in-solution hybridization enrichment method with a custom SureSelect XT kit (Agilent Technologies) and subsequent sequencingin MiSeq platform (Illumina).

The custom Sure Select oligonucleotide probe library wasdesigned including all transcripts from each target gene.Sequence capture, enrichment, and elution were performedaccording to the manufacturer’s instructions. Captured frag-ments were sequenced in pair-end 100-base mode usingMiseq (Illumina) platform. Image analysis and process-ing of the fluorescence intensities in sequences (“BaseCalling”) was performed with Real Time Analysis (RTA)software 1.8.70 (Illumina), and quality control of the datawas developed with FastQC v0.10.1 program (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). Reads werealigned to the reference genome GRCh37 with BWAv0.7.5a software (Li and Durbin, 2009). NGSrich v0.7.5software (http://ngsrich.sourceforge.net) used as a con-trol previous to variant detection, and BEDTools 2.17.0(http://bedtools.readthedocs.org/en/latest/#) and Picard 1.93(http://picard.sourceforge.net) for intermediate steps. Varscanand SAMtools v0.1.19 (Li et al., 2009) were the variant detectionsoftware used and Annovar Nov2011 (Wang et al., 2010) forvariant annotation. The assay reached a mean coverage for theGFM1 gene of 240X and 313X for exons 12 and 16 respectively.

Sanger sequencing was used to confirm the mutations iden-tified in the proband and determine phase. Exons 12 and 16 ofGFM1 gene (NM_024996) were amplified with specific primersdesigned using the free software Primer3 v.0.4.0 (www.frodo.wi.mit.edu/) and the amplified fragments sequenced using standardmethodologies. Sequencing reactions consisted of 1.0µl of previ-ously purified PCR products (ExoSAP-IT, USB, Cleveland, OH),1µl of each primer, and 1µl of Big Dye Terminator v3.1 fromthe Cycle Sequencing kit (Applied Biosystems, Foster City, CA).The reactions were run on an ABI 3730 DNA Analyzer (AppliedBiosystems, Foster City, CA). Analysis was performed with theStaden package free software.

Cell Preparation and Western Blot AnalysisHuman fibroblasts were trypsinized, pelleted and resuspended incell lysis buffer [50mM Tris-HCl pH 7.5, 130mM NaCl, 2mMMgCl2, 1% Nonidet P-40, 1mM PMSF and protease inhibitorcocktail (Roche)]. Cell lysates were vortexed briefly, centrifugedat 500 g for 5min at 4◦C and the supernatant retained.

Mitochondrial fractions were prepared from cell pellets resus-pended in homogenization buffer (0.6M mannitol, 10mM Tris-HCl pH7.4, 1mM EGTA, 0.1% BSA, and 1mM PMSF) and handhomogenized by 15 passes in a Teflon:glass dounce homogenizer.Following centrifugation at 400 g the supernatant was retainedand the pellet was re-homogenized and centrifuged as before. Thecombined supernatants were cleared at 400 g and then the crudemitochondria pelleted at 11,000 g.

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Cell lysates and mitochondrial lysates were incubatedwith sample dissociation buffer (final concentrations: 6.25mMTris/HCl pH 6.8, 2% SDS, 10% glycerol 0.01% bromophenol blueand100mM DTT) for 30min at 37◦C, separated by 12% SDS–PAGE and immobilized by wet transfer (100V, 1 h at 4◦C) onto PVDF membrane (Immobilon-P, Millipore Corporation) in25mM Tris, 192mM glycine, 0.02% SDS, and 15% methanol.Proteins of interest were bound by overnight incubation at 4◦Cwith antibodies against COXI (Abcam ab14705), SDHA (Abcamab14715), Porin/VDAC1 (Abcam ab14734), mtEFG1 (Abcamab173529), and NDUFB8 (Abcam ab110242) followed by HRP-conjugated secondary antibodies (Dako Cytomation) and visual-ized using ECL-prime (GE Healthcare) and BioRad ChemiDocMP with Image Lab software.

De novo Mitochondrial Protein SynthesisMitochondrial protein synthesis in cultured cells was performedessentially as described previously (Chomyn, 1996). Fibroblastswere labeled for 1 h in methionine/cysteine free DMEM (Sigma)with 200 uCi/ml of a [35S]-methionine/cysteine mixture (PerkinElmer) and 100 ug/ml emetine dihydrochloride (Sigma) followedby 10min chase in standard DMEM with additional 7.5 ug/mlcold methionine. Aliquots (50 ug) of total cell protein wereseparated by 15% SDS–PAGE. Signals were detected using theTyphoon FLA9500 Phosphorimager and ImageQuant software(GE Healthcare).

Genetic, Pathological, and Biochemical ResultsGenetic studies were performed sequentially in accordancewith clinical suspicion and showed a 46, XX normal kary-otype, fluorescent in situ hybridization (FISH) excluded

Wolf-Hirschhorn syndrome, direct sequencing of theFOXG1 gene was normal, multiplex ligation-dependentprobe amplification (MLPA) for subtelomeric deletionsand for CDKL5, NTNG1, and ARX genes was unremark-able. The activity of pyruvate dehydrogenase was normal infibroblasts.

Muscle histology and histochemistry showed no evidence ofobvious subsarcolemmal mitochondrial accumulation (ragged-red fibers) or inclusions and mild variation in fiber size(Figure 2A). There was a generalized decrease in histochemi-cal cytochrome c oxidase (COX) activity including a popula-tion of fibers (mosaic) that were COX-deficient (Figure 2B). Thesuccinate dehydrogenase (SDH) reaction showed a populationof pale fibers (Figure 2C), whereas the sequential COX/SDHreaction highlighted a population of weakly-reacting blue (COX-deficient) fibers (Figure 2D). In addition, the size of intracel-lular lipid droplets stained with Sudan Black appeared to beincreased (not shown). Biochemical assessment of respiratorychain enzymatic function in fibroblasts confirmed impaired com-plex I (0.019 units; controls (mean ± SD) 0.197 ± 0.034), com-plex III (0.211 units; controls (mean ± SD) 0.646 ± 0.192),and complex IV (0.165 units; controls (mean ± SD) 1.083 ±

0.186) activities, with a low complex I:II ratio (0.071; range0.58–0.90) consistent with a generalized defect of mitochon-drial translation (Figure 2E). The activity of complex V was notassessed.

Mitochondrial DNA copy number abnormalities and large-scale rearrangements were excluded in muscle; completesequencing of the mitochondrial genome in this tissue identifiedseveral well-characterized polymorphic variants, but no plausiblepathogenic mtDNA mutation.

FIGURE 2 | Muscle histology and histochemistry reveals

mitochondrial abnormalities. (A) Haematoxylin and Eosin (H&E)

histology shows mild variation in fiber size. (B) Cytochrome c oxidase

(COX) histocytochemical activity revealed a mosaic pattern of COX

deficiency without subsarcolemmal mitochondrial aggregates. (C)

Succinate dehydrogenase (SDH) reaction indicates a population of pale

fibers within the section. (D) Sequential COX-SDH histochemistry shows a

population of weak “blue” fibers corresponding to the COX-deficient,

SDH-positive fibers detected following the individual enzyme reactions. (E)

The assessment of individual respiratory chain enzyme activities in

fibroblasts identified a severe OXPHOS deficiency affecting complex I, III,

and IV in the patient (blue) compared with controls (red). Mean enzyme

activities of control fibroblasts (n = 8) are set at 100% and error bars

represent the standard deviation. *denotes values outside of the normal

range. (F) Pedigree and sequence analysis of the two mutations identified

in the patient.

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Skeletal muscle DNA was used to perform genetic screen-ing for mitochondrial encephalopathy, with simultaneous nextgeneration sequencing of the coding regions of 150 genes asso-ciated with mitochondrial respiratory chain disorders. Thisrevealed two inherited, likely pathogenic heterozygous muta-tions: c.1404delA, p.(Gly469Valfs∗84), in exon 12 (NM_024996.5,OMIM 606639) and c.2011C>T, p.(Arg671Cys), in exon 16(NM_024996.5, OMIM 606639) of the GFM1 gene (Figure 2F).The former predicts a novel frameshift mutation, whilst the lat-ter is a reported missense mutation predicting the substitutionp.(Arg671Cys) of a conserved amino acid (Calvo et al., 2012;Galmiche et al., 2012). Further analysis verified that the motherwas a heterozygous carrier of the c.1404delA p.(Gly469Valfs∗84)variant whereas the father was a heterozygous carrier of thec.2011C>T p.(Arg671Cys) variant.

Western blot analysis showed that steady state levels ofmtEFG1 protein were severely decreased in isolated mitochon-dria from patient fibroblasts (Figure 3A) compared to controlfibroblasts, confirming pathogenicity of the GFM1 mutations.Western blot analysis of patient fibroblasts showed a reduction inthe steady-state levels of NDUFB8 (complex I subunit) and COXI(complex IV subunit), but similar levels of SHDA (complex II)compared to controls (Figure 3B), which corresponds well withthe complex activity data (Figure 2E). A generalized impairment

of de novo mitochondrial protein synthesis was also observedin patient fibroblasts (Figure 3C), consistent with a decrease intranslation elongation due to the reduction of mtEFG1 proteinlevels.

Discussion

The nuclear gene, GFM1, is located at 3q25.1–q26.2 and encodesthe mitochondrial translation factor mtEFG1 (Gao et al., 2001),which acts as a five domain GTPase (Valente et al., 2007; Balasub-ramaniam et al., 2012). It catalyzes the translocation of peptidyl-tRNA from the ribosomal acceptor aminoacyl site to the peptidylsite following peptide bond formation, with the concomitantremoval of the deacylated tRNA, advancement of the mRNA byone codon and exposure of the next codon (Smits et al., 2010).

Review of Similar CasesFollowing the first report of recessive mutations in this genecausing early-onset mitochondrial disease and a generalized dis-order of mitochondrial translation (Coenen et al., 2004), 11 clin-ical cases have been published in the literature (Coenen et al.,2004; Antonicka et al., 2006; Valente et al., 2007; Smits et al., 2011;Balasubramaniam et al., 2012; Calvo et al., 2012; Galmiche et al.,

FIGURE 3 | Biochemical assessment of patient fibroblasts. (A)

Mitochondria isolated from control (C1) and patient (P) fibroblasts were

subjected to SDS-PAGE and western blot analysis using an anti-mtEFG1

antibody. Anti-porin (VDAC1) antibody was used as a loading control. (B)

Whole cell lysate from control (C1 and C2) and patient (P) primary fibroblasts

were subjected to western blot analysis. Antibodies against COXI

(mitochondrial encoded subunit of Complex IV), NDUFB8 (nuclear encoded

subunit of Complex I), SDHA (nuclear encoded subunit of Complex II) and

porin/VDAC1 (mitochondrial loading control). (C) Control (C1 and C2) and

patient (P) fibroblasts were treated with emetine dihydrochloride to inhibit

cytosolic translation and mitochondrial protein synthesis analyzed by [35S]

met/cys incorporation (1 h followed by a 10min chase). Cell lysate (50µg)

was separated through a 15% polyacrylamide gel. The gel was stained with

Coomassie blue (CBB) to confirm equal loading. Post fixation and drying the

signal was visualized by Typhoon FLA9500 PhosphorImaging. Signals were

ascribed following established migration patterns (Chomyn, 1996).

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2012). A review of the clinical, laboratory and radiological char-acteristics of the reported cases is shown in Table 1, highlightinga number of similarities between our patient and those previouslycharacterized, although there are also some notable differences.

Similar to other described cases, our patient presented withearly-onset failure to thrive, as well as encephalopathy and ele-vated blood lactate (Table 1). This latter biochemical finding wasthe only feature persistently evident in all previous patients withthe exception of those reported by Calvo and colleagues who didnot mention biochemical findings (Calvo et al., 2012). Smits andcolleagues suggested that blood lactate levels correlated with dis-ease severity (Smits et al., 2011), but this was not consistent in allthe patients described. Our patient showed a maximum plasmalactate value of 4.87mmol/L with initial responsive acidosis,whilst in other cases this ranged from 3.0 to 17mmol/L with somechildren presenting with refractory fatal lactic acidemia.

Other common features were hypo- or hypertonia, develop-mental delay, seizures in three cases (Valente et al., 2007; Smitset al., 2011; Galmiche et al., 2012) and polyneuropathy (Galmicheet al., 2012). The early onset, rapidly progressive and fatal clinicalcourse described in previously reported cases (Table 1) did notunfold in the patient we present, contrary to the hypothesis ofTrivigno and Haerry (2011). In fact, having stabilized both clini-cally and biochemically, she now maintains a reasonable qualityof life and shows no signs of imminent deterioration. However,despite her apparent clinical stability, there is some neurora-diological evidence that her condition continues to be slowlyprogressive (Figures 1E–F).

Microcephaly and minor dysmorphic traits, though notuncommon in early onset mitochondrial disease, are notuniformly described in this condition. Common neurora-diological features in the reported cases include corpus

TABLE 1 | Comparative study of clinical, laboratory and radiological findings of the patients reported with GFM1 mutations.

Clinical Reported cases

FeaturesAa Ba Cb Db Ec Fd Ge Hf If Jg Kg Lh

Consanguinity + + − − − + − + + − − −

IUGR + + + + − + + + − na na −

Microcephaly + − + + + + + + + na na −

Dysmorphism − − + − + − + + + na na −

Feeding difficulties − − − na + + − + − na na +

Seizures − − − na + + − + − na + +

Delayed growth + + + na + − + − − + na +

Tonus disturbance + + − + + + + + + + + +

D. delay + + − na + + + + + na + +

Encephalopathy + + + na + + − + − + + +

Liver failure + + + + − − + − + na na −

Myopathy − − − − − − − + − na na −

Lactate ↑ + + + + + + + + + na na +

OXPHOS ↓ Fib IV, I,(III, V) I, IV I, IV,(III, V) IV,(I, III, V) I,(III−V) I, III, IV IV, (I) I, IV I, IV IV * I, III, IV

Mu I, IV − IV, V, (I) − I, (IV) III − IV IV IV * na

CNS CC + + + − − + − − na na na +

Findings WM + + − − + + + + na na na +

BG + − − − + − + + na na na +

BS − − − − + − − + na na na −

Age - onset 10d 7w 1d In utero 3w 2d 2d 1d 2d <1w <1y 2m

Age–death 27d 5m 9d <1d 16m 2y 8m 4y 20m na na -

aCoenen et al., 2004.bAntonicka et al., 2006.cValente et al., 2007.dSmits et al., 2011.eBalasubramaniam et al., 2012.fGalmiche et al., 2012.gCalvo et al., 2012.hthis report.*Described as “Combined OXPHOS deficiencies,” but respiratory chain enzyme analysis was only reported for liver cells (marked deficient complex I and IV activities).

BG, Basal Ganglia; BS, Brainstem; CC, Corpus Callosum; CNS, Central Nervous System; d, days; D, Developmental; Fib, Fibroblasts; IU, Intrauterine; m, months old; Mu, Muscle; na,

not applicable/not available; OXPHOS, Oxidative Phosphorylation; IUGR, intrauterine growth restriction; y, years old; w, weeks old; WM, White Matter; +, present; −, absent.

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callosum thinning, leukodystrophy and basal ganglia involve-ment (Table 1), findings also identified in the patient we report.Cardiac function was preserved in all patients. The detailed clini-cal picture and outcome of the two unrelated patients mentionedby Calvo and colleagues were not thouroughly described in theirreport (Calvo et al., 2012), precluding a direct comparison atpresent.

Our patient is compound heterozygous for a novel frameshiftmutation and a reported missense mutation, resulting in asevere reduction in steady-state levels of mtEFG1 protein. Thec.1404delA, p.(Gly469Valfs∗84)mutation has not previously beendescribed, and introduces a premature termination codon intothe GFM1 transcript. Although not experimentally-confirmed,this mutation is defined as pathogenic and is highly likelyto lead to nonsense-mediated mRNA decay. The c.2011C>T,p.(Arg671Cys) pathogenic mutation has previously been iden-tified in the homozygous state in a patient presenting with acomplex phenotype including severe encephalopathy and micro-cephaly, severely decreased complex IV activity in muscle andreduced activity of complexes I and IV in skin fibroblasts(Galmiche et al., 2012) and as a heterozygous recessive muta-tion by Calvo et al. (2012).Western blot analysis showedmtEFG1protein reduced to <1% of control values in fibroblasts fromthe patient and there was also a marked decrease in full assem-bled complexes I, IV, and V compared to normal controls. Thec.1404delA, p.(Gly469Valfs∗84) and c.2011C>T, p.(Arg671Cys)mutations therefore seem highly likely to be causative of ourpatient’s clinical presentation andmultiple respiratory chain defi-ciency, indicating a disorder of generalized mitochondrial trans-lation. A comparison with the cases reported in the literature,including two sibling pairs (Coenen et al., 2004; Antonicka et al.,2006), shows all patients have inherited GFM1 mutations asrecessive Mendelian traits, with clear loss of function allelesleading to mitochondrial translation defects with a combineddeficiency of OXPHOS components (Table 2). For all describedcases, OXPHOS anomalies were suspected histochemically bydecreased COX activity in muscle and the combined deficiencyof OXPHOS components and disorder of generalized mito-chondrial translation was confirmed biochemically (Table 1).Complex II activity was consistently spared and no quantita-tive or qualitative mtDNA anomalies were found in the patients(Galmiche et al., 2012).

Interestingly, the residual steady-state levels of mutatedmtEFG1 protein have been found to be higher in heart andskeletal muscle than in liver and fibroblasts (Antonicka et al.,2006), which might diminish the susceptibility of these tissues,as has been observed in the published cases (Coenen et al., 2004;Antonicka et al., 2006; Smits et al., 2011; Trivigno and Haerry,2011). Reports suggest that the severity of the OXPHOS defectsdue to GFM1 mutations correlates with the residual level of themutant protein in each tissue (Antonicka et al., 2006; Trivignoand Haerry, 2011; Balasubramaniam et al., 2012). The differencesin residual protein levels are potentially due to diverse regulatoryand compensatory responses of the mitochondrial translationsystem in different tissues that are currently not fully understood.Indeed, altered expression of other translation elongation fac-tors (mtEFTu and mtEFTs) has been previously demonstrated in

TABLE 2 | Summary of the mutations in GFM1 gene found in the reported

patients.

Reported GFM1 mutation Type of

cases mutation

Aa Homozygous c.521A>G p.(Asn174Ser) Missense

Ba Homozygous c.521A>G p.(Asn174Ser) Missense

Cb c.961T>C p.(Ser321Pro) +

c.1765-2_1765-1delAG p.(Gly589Profs*19)

Missense +

Frameshift

Db c. 961T>C p.(Ser321Pro) +

c.1765-2_1765-1delAG p.(Gly589Profs*19)

Missense +

Frameshift

Ec c.139C>T p.(Arg47*) + c.1487T>G

p.(Met496Arg)

Nonsense +

Missense

Fd Homozygous c.748C>T p.(Arg250Trp) Missense

Ge c.539delG p.(Gly180Alafs*11) +

c.688G>A p.(Gly230Ser)

Frameshift +

Missense

Hf Homozygous c.2011C>T p.(Arg671Cys) Missense

If Homozygous c.1193T>C

p.(Leu398Pro)

Missense

Jg c.720delT p.(Glu241Asnfs*2) +

c.2011C>T p.(Arg671Cys)

Frameshift +

Missense

Kg c.720delT p.(Glu241Asnfs*2) +

c.910A>G p.(Lys304Glu)

Frameshift +

Missense

Lh c.1404delA p.(Gly469Valfs*84) +

c.2011C>T p.(Arg671Cys)

Frameshift +

Missense

aCoenen et al., 2004.bAntonicka et al., 2006.cValente et al., 2007.dSmits et al., 2011.eBalasubramaniam et al., 2012.fGalmiche et al., 2012.gCalvo et al., 2012.hthis report.

All mutations named according to reference NM_024996.5.

different tissues from a patient withGFM1mutations (Antonickaet al., 2006). The location of the specific mutation may affect par-ticular functions and interactions of the protein, which may betissue specific. For example, Galmiche and colleagues hypothe-sized that hepatic failure observed in some cases was particularlyassociated with mutations located in the central region of themtEFG1 protein and those associated with encephalopathy werelocated in peripheral segments of the protein (Galmiche et al.,2012).

These concepts support previous clinical findings describ-ing two main phenotypes, namely hepatic (Coenen et al., 2004;Antonicka et al., 2006; Balasubramaniam et al., 2012; Galmicheet al., 2012) and neurological (Valente et al., 2007; Smits et al.,2011; Galmiche et al., 2012) (Table 1). In keeping with thishypothesis, it is reasonable to suggest that the severe and exclu-sively neurological phenotype in our patient implies that thetranslation defect was most severe in the central nervous system.

The main reported causes of death have been related torespiratory complications (secondary to neurological dysfunc-tion) and to multisystem failure secondary to severe hepaticdysfunction (Table 1). Despite the severity of the OXPHOSdefects in mitochondria and the similarity of the results to theother patients, the progression of the disease appears to be more

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Brito et al. Long surviving GFM1 mutation patient

benign in our patient. At present, she is 5 years 6 months oldand has a severe encephalopathy, but has had no additionalepisodes of acute clinical or metabolic decompensation since firstcoming to medical attention at the age of 7 months. Her gen-eral condition has stabilized, without regression, and she is ableto be fed by mouth, maintaining normal growth. The specificfunctional and morphological consequences of the GFM1 muta-tions identified may help explain the particular clinical courseobserved in this case, but there are still no definitive clini-cal, laboratory, radiological or genetic characteristics that couldbe helpful in predicting disease severity and prognosis in thiscondition.

Our understanding of the mitochondrial translation appara-tus is increasing as more proteins are being identified in thepathways of mitochondrial gene expression, but these type ofdisorders remain without gene-specific treatment, much less acure (Ribas et al., 2014; Soiferman et al., 2014; Szeto, 2014). Cur-rent investigations have evaluated the effect of small molecules- largely through the activation of the PGC1α pathway tostimulate mitochondrial biogenesis and consequently ATP syn-thesis - on improving mitochondrial function in fibroblastsof patients with combined respiratory complex disorders har-boring known mutations in various nuclear-encoded compo-nents of the mitochondrial translation machinery. One suchstudy included a patient with GFM1 mutations and showedthat the compound AICAR (5-aminoimidazole-4-carboxamideribonucleotide) increased complex IV activity in GFM1 cells,demonstrating therapeutic potential (Soiferman et al., 2014).This was one example of a specific benefit of one com-pound in a particular cell line, but no universally benefi-cial compound was found, highlighting the importance of

personalized medicine in mitochondrial disease (Soiferman et al.,2014).

Concluding Remarks

Mutations in GFM1 gene are consistently associated with a com-bined respiratory chain deficiency and persistent elevation ofblood lactate. Clinical findings are heterogeneous, but neuro-logical and hepatic involvement are both prominent features.Although the previously reported cases consisted of early onset,rapidly progressive and ultimately fatal diseases, the patientdescribed here seems to show a more benign phenotype, thoughthe pathophysiology and prognosis remain unclear. To ourknowledge, this is the first patient with recessiveGFM1mutationswho has survived beyond early childhood.

Acknowledgments

We thank the “Biobanc de l’Hospital Infantil Sant Joan de Déuper a la Investigació” integrated in the Spanish Biobank Net-work of ISCIII for access to the patient muscle sample. Studiesundertaken in Spain were supported by and by grants PI11/02350and PI14/00028 from the Spanish Ministry of Health (Fondo deInvestigación Sanitaria, Instituto de Salud Carlos III). Studies

undertaken in Newcastle upon Tyne are supported by a Well-come Trust Strategic Award (096919/Z/11/Z) (RWT), the MRCCentre for Neuromuscular Diseases (G0601943) (RM and RWT),the Lily Foundation (KT, RM, and RWT) and the UK NHSHighly Specialized “Rare Mitochondrial Disorders of Adults andChildren” Service.

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Conflict of Interest Statement: The Reviewer Veronika Boczonadi declares that,

despite being affiliated to the same institution as the authors Kyle Thompson,

Steven A. Hardy, LangpingHe, and RobertMcFarland, the review process was han-

dled objectively and no conflict of interest. The authors declare that the research

was conducted in the absence of any commercial or financial relationships that

could be construed as a potential conflict of interest.

Copyright © 2015 Brito, Thompson, Campistol, Colomer, Hardy, He, Fernández-

Marmiesse, Palacios, Jou, Jiménez-Mallebrera, Armstrong, Montero, Artuch,

Tischner, Wenz, McFarland and Taylor. This is an open-access article distributed

under the terms of the Creative Commons Attribution License (CC BY). The use,

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